REGULATORS OF TUMORIGENESIS PROJECT SUMMARY Ras GTPases represent a cancer-driving nexus that is difficult to target, underscoring the need to better characterize the spectrum of Ras-interacting biomolecules that govern its function. Using new methods designed to find previously missed interactors, recent CA142635 efforts defined the Ras RNA and protein interactomes to identify a functionally important new RNA binding capacity for Ras as well as 38 new cancer-relevant Ras-interacting proteins. Ras selectively and directly bound C/D box small non-coding RNAs snoRNAs, including SNORD50A/B, which potently inhibited Ras and was deleted at high frequency co-occurantly with RAS gene mutation in human cancer. Live-cell vicinal protein labeling followed by mass spectrometry detected protein interactions missed in prior studies, among which were mTOR and the putative palmitoyltransferase, ZDHHC5. This competing renewal will define the function of these new Ras interacting RNA and proteins in Ras-driven tumorigenesis. First, we will further characterize SNORD50A/B snoRNA impacts on Ras as well as the role of other newly discovered Ras-binding snoRNAs. Based on the finding that SNORD50A/B snoRNAs block farnesyltransferase binding to Ras, we will define the global impact of SNORD50A/B deletion on Ras protein-protein interactions to shed further light on the newly discovered modulation of Ras by RNA. We will also systematically test the function of additional Ras-binding RNAs on Ras-driven neoplasia, beginning with 6 snoRNAs that are recurrently mutated at high frequency in human cancer. Aim I will test the hypotheses that SNORD50A/B snoRNAs remodel the Ras protein interactome to regulate Ras signaling and that additional Ras-binding snoRNAs alter Ras action in tumorigenesis. Second, we will characterize the newly identified Ras-mTOR interaction in tumorigenesis and will define the functional importance of additional vicinal proteins to oncogenic Ras function. We will define the domains on Ras isoforms and mTOR responsible for the observed direct binding of Ras to mTOR, examine the impact of Ras-mTOR binding on mTORC1 vs mTORC2 kinase activity and assess the impacts of disrupting Ras-mTOR binding on Ras- driven tumorigenesis in vivo. We will also systematically delete 38 newly identified Ras proximal proteins and quantify their impact on Ras-driven tumorigenesis. Aim II will test the hypotheses that Ras-mTOR binding is functional in Ras-driven tumorigenesis and that multiple newly identified Ras-interacting proteins impact Ras signaling outcomes in neoplasia. At the end of the proposed funding cycle, this effort will define the function of newly identified Ras-binding RNAs and Ras-proximal proteins in Ras-driven tumorigenesis. 1